The separation of the DNA strands during replication and genetic recombination must involve the simultaneous unpairing of the bases in the opposing strands and the unwinding of the DNA helix. This proposal is concerned with the enzymes that are involved in these aspects of the biochemistry of DNA. One of the major objectives of this project is to identify the enzymatic machinery responsible for providing the swivels during DNA replication. We will determine whether the DNA untwisting exzyme (nicking-closing enzyme) has this role in the replication of the circular SV40 DNA. We will attempt to determine whether a DNA gyrase is present in eucaryotic cells and if so, whether it contributes to the process of helix unwinding in vivo. A related objectve is to continue our studies on the mechanism of the DNA untwisting enzyme with particular attention to the nature of the covalent linkage between the enzyme and the nicked intermediate. A second objective is to identify and purify an enzyme activity from rat liver nuclei which utilizes the hydrolysis of ATP to drive the unwinding of the DNA helix. We will characterize any DNA unwinding enzyme(s) we discover with respect to cofactor and substrate requirements. We will attempt to determine whether the enzyme(s) does indeed catalyze helix unwinding during DNA replication and/or genetic recombination.